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Rationale: Busulfan is currently an indispensable anti-cancer drug, particularly for children, but the side effects on male reproduction are so serious that critical drug management is needed to minimize any negative impact. Meanwhile, alginate oligosaccharides (AOS) are natural products with many consequent advantages, that have attracted a great deal of pharmaceutical attention. In the current investigation, we performed single-cell RNA sequencing on murine testes treated with busulfan and/or AOS to define the mitigating effects of AOS on spermatogenesis at the single cell level. Methods: Testicular cells (in vivo) were examined by single cell RNA sequencing analysis, histopathological analysis, immunofluorescence staining, and Western blotting. Testes samples (ex vivo) underwent RNA sequencing analysis. Blood and testicular metabolomes were determined by liquid chromatography-mass spectrometry (LC/MS). Results: We found that AOS increased murine sperm concentration and motility, and rescued busulfan disrupted spermatogenesis through improving (i) the proportion of germ cells, (ii) gene expression important for spermatogenesis, and (iii) transcriptional factors in vivo. Furthermore, AOS promoted the ex vivo expression of genes important for spermatogenesis. Finally, our results showed that AOS improved blood and testis metabolomes as well as the gut microbiota to support the recovery of spermatogenesis. Conclusions: AOS could be used to improve fertility in patients undergoing chemotherapy and to combat other factors that induce infertility in humans.

Jasmonic acid (JA) is involved in a variety of physiological responses in seed plants. However, the detection and role of JA in lycophytes, a group of seedless vascular plants, have remained elusive until recently. This study provides the first evidence of 12-oxo-phytodienoic acid (OPDA), JA and jasmonoyl-isoleucine (JA-Ile) in the model lycophyte Selaginella moellendorffii. Mechanical wounding stimulated the accumulation of OPDA, JA and JA-Ile. These data were corroborated by the detection of enzymatically active allene oxide synthase (AOS), allene oxide cyclase (AOC), 12-oxo-phytodienoic acid reductase 3 (OPR3) and JA-Ile synthase (JAR1) in S. moellendorffii. SmAOS2 is involved in the first committed step of JA biosynthesis. SmAOC1 is a crucial enzyme for generating the basic structure of jasmonates and is actively involved in the formation of OPDA. SmOPR5, a functionally active OPR3-like enzyme, is also vital for the reduction of (+)-cis-OPDA, the only isomer of the JA precursor. The conjugation of JA to Ile by SmJAR1 demonstrates that S. moellendorffii produces JA-Ile. Thus, the four active enzymes have characteristics similar to those in seed plants. Wounding and JA treatment induced the expression of SmAOC1 and SmOPR5. Furthermore, JA inhibited the growth of shoots in S. moellendorffii, which suggests that JA functions as a signaling molecule in S. moellendorffii. This study proposes that JA evolved as a plant hormone for stress adaptation, beginning with the emergence of vascular plants.

The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239T was generated, revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences (CDSs). Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17T, Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures, including various genome sizes, core/quasi-core/non-core configurations and different kinds of episomes. Although polyketide synthase gene clusters were absent from the A. methanolica genome, 12 gene clusters related to the biosynthesis of other specialized (secondary) metabolites were identified. Complete pathways attributable to the facultative methylotrophic physiology of A. methanolica strain 239T, including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle, were identified together with evidence that the latter might be the result of horizontal gene transfer. Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452, a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains, respectively, revealed three major phyletic lineages, namely the mesophilic or moderately thermophilic A. orientalis subclade (AOS), the mesophilic Amycolatopsis taiwanensis subclade (ATS) and the thermophilic A. methanolica subclade (AMS). The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes. This study shows the value of integrating conventional taxonomic with whole genome sequence data.

Background Schistosomiasis, one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers (ALP; morphological characteristics of egg; CalS (calcified substance); AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers (CalS; AOS; SDHG and SjR2 mRNA.), to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis. Methods Tissue samples were collected from mice at six different time points during S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP (alkaline phosphatase) levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability. Results The results of the study showed that the morphology of S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers (CalS, AOS, SDHG and SjR2 mRNA) were morphologically difficult to identify. Conclusions Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2362-4) contains supplementary material, which is available to authorized users.

To better understand the physiological role of jasmonates (JAs) during fruit development and ripening, and relationships with ethylene, the time course of transcript accumulation of the key enzyme in JA biosynthesis, allene oxide synthase (AOS), was analyzed during S1–S4 fruit developmental stages, until ripening, in the mesocarp and seed of peach ( Prunus persica var. laevis Gray), cv. ‘Stark Red Gold’. In the mesocarp, transcript amounts of PpAOS1 were the highest in S1–S2, then decreased to almost undetectable levels at the transition S3/S4, to rise slightly at harvest. In parallel, JA concentration was measured in the mesocarp during development; maximum jasmonic acid accumulation was observed in S1, the lowest levels in S2 and S3 and a new rise in S4. In the seed, an AOS1 transcript was detected whose accumulation differed in the embryo as compared to cotyledons during seed growth. The reciprocal JA/ethylene relationships were investigated in fruit at different physiological stages by exogenously supplying methyl jasmonate (MJ). The effects of treatments with 0.88 mM MJ on transcript levels of PpAOS1 and ethylene biosynthetic genes PpACS1 , PpACO1 and PpACO2 were evaluated in the mesocarp and seed of detached nectarines at an early (S1) and late (S4) developmental stage. Results show that JA biosynthesis appears to be developmentally regulated in seed and mesocarp of nectarine fruit and that the response to MJ depends upon the fruit physiological stage.

Elicitors/pathogen-associated molecular patterns (PAMPs) trigger the plant immune system, leading to rapid programmed cell death (hypersensitive response, HR) and stomatal closure. Previous reports have shown that the vacuolar processing enzyme (VPE), a cysteine proteinase responsible for the maturation of vacuolar proteins, has caspase-1-like activity and mediates TMV- and mycotoxin-induced cell death. The role of VPE from Nicotiana benthamiana in the response to three elicitors: bacterial harpin, fungal Nep1, and oomycete boehmerin, is described here. Single-silenced (NbVPE1a or NbVPE1b) and dual-silenced (NbVPE1a/1b) N. benthamiana plants were produced by virus-induced gene silencing. Although NbVPE silencing does not affect H(2)O(2) accumulation triggered by boehmerin, harpin, or Nep1, the HR is absent in NbVPE1a- and NbVPE1a/1b-silenced plants treated with harpin alone. However, NbVPE-silenced plants develop a normal HR after boehmerin and Nep1 treatment. These results suggest that harpin-triggered HR is VPE-dependent. Surprisingly, all gene-silenced plants show significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells. Dual-silenced plants show increased elicitor-triggered AOS production in guard cells. The accumulation of transcripts associated with defence and cell redox is modified by VPE silencing in elicitor signalling. Overall, these results indicate that VPE from N. benthamiana functions not only in elicitor-induced HR, but also in elicitor-induced stomatal closure, suggesting that VPE may be involved in elicitor-triggered immunity.

Active oxygen species (AOS) are central components of the defence reactions of plants against pathogens. Plant respiratory burst oxidase homologues (RBOH) of gp91(phox), a plasma membrane protein of the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, play a prominent role in AOS production. The role of two RBOH from Nicotiana benthamiana, NbrbohA and NbrbohB that encode plant NADPH oxidase in the process of elicitor-induced stomatal closure and hypersensitive cell death is described here. NbrbohA was constitutively expressed at a low level, whereas NbrbohB was induced when protein elicitors exist (such as boehmerin, harpin, or INF1). The virus-induced gene-silencing (VIGS) method was used to produce single-silenced (NbrbohA or NbrbohB) and double-silenced (NbrbohA and NbrbohB) N. benthamiana plants. The hypersensitive response (HR) of cell death and pathogenesis-related (PR) gene expression of these gene-silenced N. benthamiana plants, induced by various elicitors, are examined. The HR cell death and transcript accumulation of genes related to the defence response (PR1) were slightly affected, suggesting that RBOH are not essential for elicitor-induced HR and activation of these genes. Interestingly, gene-silenced plants impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production, but not elicitor-induced cytosolic calcium ion accumulation and elicitor-triggered AOS production in guard cells. These results indicate that RBOH from N. benthamiana function in elicitor-induced stomatal closure, but not in elicitor-induced HR.

Background: Dietary supplementation with oligosaccharides has been proven to be beneficial for health in several mammalian species. Next to prebiotic effects resulting in a modulation of gut micro biota, immunomodulatory effects of oligosaccharides have been documented in vivo. Supplementation with defined oligosaccharide fractions has been shown to attenuate allergic responses and enhance defensive immune responses. Despite the accumulating evidence for immunomodulatory effects, very limited information is available regarding the direct mechanism of action of oligosaccharides. This study aims to elucidate the effects of selected oligosaccharide fractions on the lipopolysaccharide (LPS) induced inflammatory response in equine peripheral blood mononuclear cells (PBMCs). We investigated three different products containing either galacto-oligosaccharides (GOS) alone, a combination of GOS with fructo-oligosaccharides (FOS), and a triple combination of GOS and FOS with acidic oligosaccharides (AOS), at different concentrations. These products have been used in an identical composition in various previously published in vivo experiments. As the selected oligosaccharide fractions were derived from natural products, the fractions contained defined amounts of mono- and disaccharides and minor amounts of endotoxin, which was taken into account in the design of the study and the analysis of data. Acquired data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. Results: Exposing cultured PBMCs to either GOS or GOS/FOS fractions resulted in a substantial dose-dependent increase of tumour necrosis factor-α (TNF-α) production in LPS challenged PBMCs. In contrast, incubation with GOS/FOS/AOS resulted in a dose-dependent reduction of both TNF-α and interleukin-10 production following LPS challenge. In addition, incubation with GOS/FOS/AOS significantly increased the apparent PBMC viability, indicating a protective or mitogenic effect. Furthermore, mono- and disaccharide control fractions significantly stimulated the inflammatory response in LPS challenged PBMCs as well, though to a lesser extent than GOS and GOS/FOS fractions. Conclusions: We found distinct immunomodulating effects of the investigated standardised oligosaccharide fractions, which either stimulated or suppressed the LPS induced inflammatory response in PBMCs. Both scenarios require additional investigation, to elucidate underlying modulatory mechanisms, and to translate this knowledge into the clinical application of oligosaccharide supplements in foals and other neonates.

Heterotrimeric G-proteins have been implicated in having a role in many plant signalling pathways. To understand further the role of G-proteins, a preliminary experiment was performed to assess the impact of the Gα subunit loss-of-function mutation gpa1-1 on the Arabidopsis transcriptome. The analysis indicated that the Gα subunit may play a role in response to jasmonic acid (JA). Consistent with this, Gα mutants showed a reduced response to JA in inhibition of chlorophyll accumulation and root growth, whilst Gα gain-of-function plants overexpressing Gα showed the opposite phenotype. The levels of JA and related compounds were unaffected in the gpa1-1 mutant, as was autoregulation of the Allene Oxide Synthase (AOS) gene that encodes a key enzyme for JA biosynthesis. In contrast, further analyses using Gα loss- and gain-of-function Arabidopsis lines indicated that Gα positively modulates the expression of the Vegetative Storage Protein (VSP) gene. This indicates that the Gα subunit regulates a subset of JA-regulated genes defining a branch point in this signalling pathway in Arabidopsis. Further analysis of the impact of Gα loss of function upon the JA-regulated transcriptome using Arabidopsis full genome arrays indicated that up to 29% of genes that are >2-fold regulated by JA in the wild type are misregulated in the Gα mutant. This supports the observation that a significant proportion of, but not all, JA-regulated gene expression is mediated by Gα.

Summary A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91–phox in AOS production in elicited tobacco cells.